Macrophages can promote the growth of some tumors, such as those of the breast and lung, but it is unknown whether this is true for all tumors, including those of the nervous system. On the contrary, we have previously shown that macrophages can slow the progression of malignant gliomas through a tumor necrosis factor-dependent mechanism. Here, we provide evidence suggesting that this antitumor effect could be mediated by T lymphocytes, as their number was drastically reduced in tumor necrosis factor-deficient mice and inversely correlated with glioma volume. However, this correlation was only observed in allogeneic recipients, prompting a reevaluation of the role of macrophages in a nonimmunogenic context. Using syngeneic mice expressing the herpes simplex virus thymidine kinase under the control of the CD11b promoter, we show that macrophages can exert an antitumor effect without the help of T lymphocytes. Macrophage depletion achieved by ganciclovir treatment resulted in a 33% increase in glioma volume. The antitumor effect of macrophages was not likely due to a tumoricidal activity because phagocytosis or apoptosis of glioma cells, transduced ex vivo with a lentiviral vector expressing green fluorescent protein, was rarely observed. Their antitumor effect was also not due to a destructive action on the tumor vasculature because macrophage depletion resulted in a modest reduction in vascular density. Therefore, this study suggests that macrophages can attenuate glioma growth by an unconventional mechanism. This study also validates a new transgenic model to explore the role of macrophages in cancer.